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The occurrence of antibacterial resistance has become a global concern. The results of previous studies revealed many cases of antibacterial resistance of levofloxacin in H. pylori strains originating from Indonesia. Conventional attempts to obtain new antibacterial agents usually take a long time, other efforts are needed to speed up discovery. Databases that provide the structure of DNA gyrases cannot fully provide what is needed. This study aims to obtain the three-dimensional structure of DNA gyrase of the Indonesian H. pylori strain so that it can be used to simulate the interaction process of compounds that have the potential to be anti-H. pylori through in silico studies. This study used in silico methods to predict the absorption, distribution, metabolism, excretion, and toxicology of potential compounds. The next process involves screening for drug similarity based on criteria set by Muegge, Egan, Veber, Lipinski, and Ghose. The three-dimensional structure of DNA gyrase is formed from several samples of amino acid sequences from the Indonesian gyrA and gyrB H. pylori strains. Eligible potential compounds were subjected to molecular docking studies on gyrA and gyrB to obtain the best score. The three-dimensional structure of the Indonesian gyrA and gyrB H. pylori strains has been obtained. Eugenol, cinnamaldehyde, and ethyl p-methoxycinnamate were more easily absorbed compared to curcumin and levofloxacin as controls. Curcumin and hesperidin show poor absorption and longer distribution in the gastric mucosa, thus allowing long-term interaction with H. pylori. All compounds do not inhibit cytochrome P450, can be excreted by the body, and are non-toxic. The prediction of drug similarity passes only through curcumin and ethyl p-methoxycinnamate. The results of molecular docking concluded that curcumin and hesperidin had better scores than levofloxacin. Curcumin has the most potential as an anti-H. pylori. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, a chronic granulomatous enteritis leading to economic losses and posing a risk to human health due to its zoonotic potential. The pathogen cannot reliably be detected by standard methods, and immunological procedures during the infection are not well understood. Therefore, the aim of our study was to explore host–pathogen interactions in MAP-infected dairy cows and to improve diagnostic tests. Serum proteomics analysis using quantitative label-free LC-MS/MS revealed 60 differentially abundant proteins in MAP-infected dairy cows compared to healthy controls from the same infected herd and 90 differentially abundant proteins in comparison to another control group from an uninfected herd. Pathway enrichment analysis provided new insights into the immune response to MAP and susceptibility to the infection. Furthermore, we found a higher abundance of Cathepsin S (CTSS) in the serum of MAP-infected dairy cows, which is involved in multiple enriched pathways associated with the immune system. Confirmed with Western blotting, we identified CTSS as a potential biomarker for bovine paratuberculosis. This study enabled a better understanding of procedures in the host–pathogen response to MAP and improved detection of paratuberculosis-diseased cattle. [ABSTRACT FROM AUTHOR]

Simple Summary: Paratuberculosis impacts animal welfare and the economic performance of dairy herds by causing reduced milk yield and carcass weight and premature culling. Due to the ability of the infectious agent Mycobacterium avium subsp. paratuberculosis (MAP) to survive in the environment for a long time, the limited diagnostic accuracy of tests, and the long incubation period, successful elimination of MAP from a cattle herd is doubted. This study describes measures applied to control the disease in a 450-head dairy herd and, as the result of these measures, the progress of prevalence reduction to a level where the infectious agent was not detectable anymore in any individual sample and from the liquid manure of the herd. MAP shedders were detected by the bacteriological cultivation of individual faecal samples from each cow in annual intervals and MAP shedders were removed from the herd in a timely manner. Calves were kept outside the barn of adult cows and hygienic measures to minimize their MAP exposure were established. The results demonstrate that the elimination of MAP might be possible within 10 years. Voluntary control programs are effective in controlling paratuberculosis in closed herds by providing adequate diagnostic, logistic, and financial support for farmers. This longitudinal case study provides an in-detail report of the process towards the elimination of Mycobacterium avium subsp. paratuberculosis (MAP) from a closed 450-head commercial dairy herd. In parallel, two diagnostic approaches were applied to all cows in annual intervals during 2012–2022: detection of MAP in individual faecal samples by bacteriological cultivation on solid medium and detection of MAP-specific antibodies by ELISA. For each annual sampling, the kappa coefficients for test agreement and the survival rates of MAP-positive and MAP-negative cows were calculated. Applying a multivariable linear regression model revealed a significantly lower fat-corrected 305-day milk yield for MAP-positive cows. The true prevalence of MAP shedders reduced from 24.2% in 2012 to 0.4% in 2019 and during 2020–2022, no MAP shedder was identified. Test agreement was generally low and bacteriological cultivation showed positive results earlier than the ELISA. In the first years of control, the survival of MAP shedders was longer than in the final stage. In conclusion, the elimination of MAP from a dairy herd might be feasible within a decade. Changes in the test agreement must be considered. Timely removal of MAP shedders, hygienic calf rearing, and colostrum supply are key for successful control. [ABSTRACT FROM AUTHOR]

Vaccination is the most effective tool for paratuberculosis control. Currently, available vaccines prevent the progression of clinical disease in most animals but do not fully protect them against infection and induce the formation of an injection site granuloma. The precise mechanisms that operate in response to vaccination and granuloma development, as well as the effect that adjuvants could trigger, have not been fully investigated. Therefore, this study aimed to investigate the injection site granulomas induced by two inactivated paratuberculosis vaccines, which differ in the adjuvant employed. Two groups of 45-day-old lambs were immunized with two commercially available vaccines--one (n = 4) with Gudair® and the other (n = 4) with Silirum®. A third group (n = 4) was not vaccinated and served as control. The peripheral humoral response was assessed throughout the study by a commercial anti- Mycobacterium avium subspecies paratuberculosis (Map) antibody indirect ELISA, and the cellular immune response was assessed similarly by the IFN-g release and comparative intradermal tests. The injection site granulomas were measured during the experiment and sampled at 75 days post-vaccination (dpv) when the animals were euthanized. The tissue damage, antigen and adjuvant distribution, and the presence and amount of immune cells were then determined and assessed by immunohistochemicalmethods. Antibodies against Map antigens; a general macrophage marker (Iba1), M1 (iNOS), and M2 (CD204) macrophages; T (CD3), B (CD20), and gd T lymphocytes, proteins MHC-II and NRAMP1, and cytokines IL-4, IL-10, TNF, and IFN-g were employed. Silirum® elicited a stronger peripheral cellular immune response than Gudair®, while the latter induced larger granulomas and more tissue damage at the site of injection. Additionally, adjuvant and Map antigen distribution throughout the granulomatous inflammatory infiltrate, as well as the NRAMP1 cell expression, which is linked to antigen phagocytosis, were highly irregular. In Silirum® induced granulomas, a higher number of MHC-II and TNF-expressing cells and a lower number of M2 macrophages suggested an improved antigen presentation, which could be due to the better antigen distribution and reduced tissue damage induced by this vaccine. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of Johne's disease (JD) in ruminants, establishes a prolonged and often lifelong enteric infection. The implementation of control measures for bovine JD has faced obstacles due to the considerable expenses involved in disease surveillance and hindered by unreliable and inadequate diagnostic tests, emphasizing the need for an effective vaccine that can stimulate mucosal immunity in the gastrointestinal tract. Previous investigations have demonstrated that deletion of the BacA gene in MAP produces an attenuated strain that can transiently colonize the calf small intestine while retaining its capacity to stimulate systemic immune responses similar to wildtype MAP strains. This study assessed the efficacy of the BacA gene deletion MAP strain, referred to as the BacA vaccine, when administered orally to young calves. The research aimed to evaluate its effectiveness in controlling MAP intestinal infection and to investigate the immune responses elicited by mucosal vaccination. The study represents the first evaluation of an enteric modified live MAP vaccine in the context of an oral MAP challenge in young calves. Oral immunization with BacA reduced MAP colonization specifically in the ileum and ileocecal valve. This partially protective immune response was associated with an increased frequency of CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFNγ+/TNFα+) in vaccinated animals. Moreover, re-stimulated PBMCs from vaccinated animals showed increased expression of IFNγ, IP-10, IL-2, and IL-17 at 10- and 12-weeks post challenge. Furthermore, immunophenotyping of blood leukocytes revealed that vaccinated calves had increased levels of T cells expressing cell-surface markers consistent with long-term central memory. Overall, our findings provide new insights into the development and immunogenicity of a modified live MAP vaccine against bovine JD, demonstrating oral vaccination can stimulate host immune responses that can be protective against enteric MAP infection. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is an opportunistic pathogen linked to outbreaks in powdered infant formula (PIF), primarily causing meningitis and necrotizing enterocolitis. Whole-genome sequencing (WGS) was used to characterize 18 C. sakazakii strains isolated from PIF (powdered infant formula) manufacturing plants (2011–2015). Sequence Type (ST) 1 was identified as the dominant sequence type, and all isolates carried virulence genes for chemotaxis, flagellar motion, and heat shock proteins. Multiple antibiotic resistance genes were detected, with all isolates exhibiting resistance to Cephalosporins and Tetracycline. A significant correlation existed between genotypic and phenotypic antibiotic resistance. The plasmid Col(pHAD28) was identified in the isolates recovered from the same PIF environment. All isolates harbored at least one intact phage. All the study isolates were compared with a collection of 96 publicly available C. sakazakii genomes to place these isolates within a global context. This comprehensive study, integrating phylogenetic, genomic, and epidemiological data, contributes to a deeper understanding of Cronobacter outbreaks. It provides valuable insights to enhance surveillance, prevention, and control strategies in food processing and public health contexts. [ABSTRACT FROM AUTHOR]

Current management of gastric inflammation involves the eradication of Helicobacter pylori. However, the effectiveness of commonly used antibiotics against H. pylori infection has decreased due to antibiotic resistance. Phenotypic-based diagnostics are laborious and finding the cause of resistance can be difficult. Therefore, early detection and understanding of the underlying mechanism of this resistance are necessary. This study evaluated the mutations in the genes related to the Antimicrobial Resistance (AMR) of the clinical isolates from Bangladeshi subjects. Whole-genome sequencing was performed on 56 isolates and the genes (such as pbp1a, rdxA, ribF, fur, gyrA, gyrB, 23S rRNA, and infB) were extracted. The reads were assembled, and the SNPs were extracted by the latest pipeline for antibiotic mutation analysis, ARIBA. The mutations and the association with the antibiotic phenotypes were evaluated using Fisher's exact test. In this study, the clarithromycin resistance rate was high, 39.3% (22/56), with the median MIC 24 mg/L ranging from 2 to 128 mg/L. The mutation of A2147G was significantly associated with resistance (p = 0.000018) but not in locus A2146G (p = 0.056). Levofloxacin also posed a high resistance. We observed that the mutation of D91N (but not D91Y) (p = 0.002) and N87K (p = 0.002) of gyrA was associated with levofloxacin resistance. Mutations in locus A343V (p = 0.041) of gyrB also showed a significant association. Meanwhile, in the pbp1a gene, several mutations might explain the resistance; they were G594fs (p = 0.036), K306R (p = 0.036), N562Y (p = 0.0006), and V45I (p = 0.018). The prevalence of metronidazole was exceptionally high (96.4%), and numerous mutations occurred in rdxA genes, including the truncation of genes. These results imply that the mutation in genes encoding the target protein of antibiotics remains the critical resistance mechanism in H. pylori. [ABSTRACT FROM AUTHOR]

Simple Summary: This study reports the 10 years (2011–2020) follow-up of a cattle herd, with a high prevalence of paratuberculosis (PTB) cohabiting with a population of pigeons. After 6 years without tests, the herd returned to a high prevalence, and the PTB program control was revised. Likewise, the presence of MAP in feral pigeons was studied to evaluate their role as a transmitter and/or reservoir, as well as their influence on controlling the PTB. Antibodies values according to the optical density (OD) determined by ELISA increased each year and could be used to isolate herds close to the cutoff point to improve PTB control. The maintenance of ELISA tests, even though there are no positive animals, is essential for the success of the PTB control. PCR results positive against MAP samples of intestine, foot skin, and feces were observed in pigeons. The follow-up carried out in this study made it possible to establish the wild pigeons as mechanical vectors of PTB in cattle after confirmation of MAP in the foot, as well as the possibility of a possible in intestine and feces. Although future studies should be carried out to clarify the true importance of these findings in the PTB control program in cattle, the control of pigeons in farm could be important. A bovine herd with a high prevalence of paratuberculosis (PTB) cohabiting with a population of pigeons was studied (2011–2020). After finding the disease in 2011, annual monitoring was performed in 2012–2014 by obtaining blood samples for ELISA and intradermal tuberculinization (IT) tests for Mycobacterium avium subsp. paratuberculosis (MAP). Positive animals were eliminated. PTB prevalence dropped from 10% to 0% but returned to similar values (9.5%) after 6 years without tests. In all animals, Ac values according to the optical density (OD) determined by ELISA increased each year and could be used to isolate herds close to the cutoff point to improve PTB control. Possible reservoirs were considered after evaluating the little success of the PTB control program, and the population of feral pigeons was studied. Specifically, 10% of the pigeon population (n = 13) was necropsied. Samples of intestine, feces, and foot skin for PCR study for MAP and samples of terminal intestine for histopathological analysis were taken. Eleven pigeons were PCR-positive against MAP, in the intestine (10/11), foot skin (3/11), and feces (1/11). The presence of MAP in pigeon feet could demonstrate its role as a mechanical disseminator of PTB, while the presence in pigeon intestine and feces could also suggest its role as a reservoir. [ABSTRACT FROM AUTHOR]

Simple Summary: Paratuberculosis is an economically important disease due to its negative effect on animal production. One of the tools for controlling this disease is vaccination. The peripheral humoral and cellular immune responses and animal losses—regardless of reason—depending on whether or not they were vaccinated and their age, were studied for almost two years in a herd of dairy goats. The immune response was greater and more persistent when vaccination took place at an earlier age. Moreover, animals vaccinated at this time showed heterologous protection, as evidenced by a decrease in the number of dead and culled animals compared to those not vaccinated. Based on the cellular immune response, no cross-reactivity was observed when an intradermal skin test for tuberculosis diagnosis was employed. Vaccination could be considered as an effective method for paratuberculosis control, although controversial, with a need for investigation in some aspects. The objective of this study was to evaluate the effect of vaccination, depending on the age of the animals, on their immune response, the reduction of paratuberculosis cases, mortality and culled animals in a commercial dairy herd. Goats from three different ages were immunized with the inactivated Gudair® vaccine. Peripheral antibody and IFN-γ output were evaluated for 21 months post-vaccination (mpv) and intradermal skin tests (IDSTs) for tuberculosis, with avian- and bovine-purified protein derivatives (PPD), were carried out at 6 and at 18 mpv to evaluate the humoral and cellular immune peripheral responses, respectively. The number of dead or culled animals, regardless of the reason, was also monitored and the causes of death determined by pathological examination. A significant increase in the production of IFN-γ was observed in all the vaccinated groups when the blood samples were stimulated with avian PPD, from 3 mpv to 18 mpv, and with bovine PPD, between 3 and 21 mpv. Moreover, serum antibody levels increased between 3 and 21 mpv in all vaccinated groups. The highest levels were found in animals vaccinated at 5 months, and the lowest in adult individuals. No positive reactants to tuberculosis were found by intradermal skin test. No animal losses associated with clinical paratuberculosis were detected in any of the groups. The number of total culled animals was significantly lower in the vaccinated than in the unvaccinated groups, especially on 1.5-month-old vaccinated kids. These results suggest that vaccination of paratuberculosis, especially in young animals, could induce heterologous protection. [ABSTRACT FROM AUTHOR]

To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of MAP's pathogenesis and potentially develop a live attenuated Johne's disease vaccine by knocking out these genes. MAP genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A saturated library of 3,852 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body. [ABSTRACT FROM AUTHOR]

Vaccination can be an efficient method for the control of paratuberculosis in ruminants. However, the official tuberculosis control tests cross-interfere with the animals vaccinated against paratuberculosis. In order to test and compare new antigens that could solve this problem, the production of interferon-gamma (IFN-γ) in peripheral blood at different post-vaccination days in experimental kids and adult goats, in field conditions, using the avian and bovine purified protein derivative (PPD), the johnin, two peptide cocktails of Mycobacterium bovis (PC-EC and PC-HP) and the antigens VK 055 and VK 067 of Mycobacterium avium subspecies paratuberculosis (Map) has been analyzed in vitro. The non-specific production of IFN-γ was observed after blood stimulation with the PC-EC and PC-HP cocktail in any sample from vaccinated animals, whereas it was detected when bovine PPD was used. These results support the possible use of these new Mycobacterium bovis antigens in the in the differentiation of animals vaccinated against paratuberculosis or infected with tuberculosis by improving the specificity of bovine PPD. In contrast, the two Map antigens tested in this study did not improve the sensitivity of johnin or avian PPD in the detection of vaccinated or Map-infected goats. [ABSTRACT FROM AUTHOR]

The information generated from the official eradication programmes of caprine tuberculosis (TB) in Castilla y León, Spain, during 2018, has been used to assess the effect of vaccination against paratuberculosis (PTB) and the presence of this infection, on the single intradermal tuberculin (SIT) test results. Data from 121,665 goats belonging to 1936 different herds were analysed using generalized linear models. An epidemiological survey was conducted to know the herd immunization status against PTB and the date of last vaccination. All SIT test‐positive animals were further investigated in order to confirm the diagnosis of TB, through bacterial culture, and PTB, by histopathological and qPCR analyses. SIT positivity was found in 39 (2.01%) herds and 507 (0.41%) goats. TB was confirmed by M. caprae or M. bovis isolation in 10 (0.51%) herds and 46 (0.038%) goats. PTB was diagnosed in 13 (33.33%) and 55 (10.84%) of the SIT test‐positive herds and goats, respectively. Vaccination against PTB showed a significant influence on the results of the SIT test at herd level, with higher positivity detected among those herds vaccinated. However, this effect was not observed when the total number of animals was considered, where the highest positivity was found in unvaccinated goats. The time elapsed between vaccination and SIT test performance also influenced the results. The strongest effect was found when less than eight months elapsed between performing both activities, and to a lesser extent between 8 and 12 months. Conversely, no positive herds or animals were found when the time elapsed was higher than one year. No significant effect of the presence of PTB was observed. These findings demonstrate that the use of PTB vaccine does not result in false positives to a SIT test at individual level, provided that the time elapsed between the performance of both practices is higher than 12 months. [ABSTRACT FROM AUTHOR]

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102−104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns. [ABSTRACT FROM AUTHOR]

The willingness to pay (WTP) approach is increasingly being used in different disciplines to assess peoples' readiness to accept change. This paper assesses the potential for two subunit vaccines for the prevention and control of bovine tuberculosis and paratuberculosis in cattle. A survey of beef and dairy farmers was conducted across Canada to identify factors that influence their WTP for subunit vaccines. Estimated results of the interval‐data model indicate that the size of a farmer's cattle herd, neighbourhood effect, and buyer recommendations for vaccination significantly influence farmers' WTP while veterinarians appear to be the most critical pathway for farmers to source information on new vaccine options. The mean willingness to pay amounts for both vaccines reveals that farmers are likely to use the vaccines if the costs are kept at reasonable level. [ABSTRACT FROM AUTHOR]

The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated Mycobacterium avium subsp. paratuberculosis (MAP) ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands. Analyzed herds were representative for location and herd size of dairy goat herds in the Netherlands. A significantly higher proportion of the analyzed 49 herds were organic as compared with the total Dutch dairy goat population. First, the MAP ELISA was optimized using 992 paired serum and milk samples. At a cut-off of 25 S/P%, the relative sensitivity (Se) was 58.4% (n = 992, 95% CI: 48.8% 67.6%) and relative specificity (Sp) was 98.5% (n = 992, 95% CI: 97.5% 99.2%), as compared to serum ELISA results. The percentage of positively tested herds was 78.2% (n = 49, 95% CI: 63.4% 88.1%). The percentage of positive milk samples per herd (n = 22) was on average 4.6% (median, min, and max of 4.7%, 0.0%, and 10.7%, respectively). Average age of ELISA-positive (3.2 years) and -negative goats (3.2 years) was not different. Significantly more vaccinated goats tested positive (6.7%) as compared with nonvaccinated goats (1.1%). This study shows that a high number of vaccinated and nonvaccinated commercial dairy goat herds in the Netherlands have MAP-ELISA-positive goats. [ABSTRACT FROM AUTHOR]

The current study proposed that previously characterized individual antigenic proteins could represent potential replacement for conventional purified protein derivative (PPD) in tuberculosis skin testing when used in cocktails triggered by suitable TLR-stimulants that would provide the missing pro-inflammatory stimulus. Three different cocktails of previously selected antigens, including C1 (ESAT-6/CPF-10/MPB-83); C2 (ESAT-6/MPB-64/MPB-83); and C3 (CPF-10/MPB-64/MPB-83), were evaluated in vitro using lymphocytic proliferation and IFN-γ production assays, as well as mRNA and protein expression levels of TNF-α, IL-12p40, and IL-2 as pro-inflammatory molecules. C1 showed the highest significant induction of pro-inflammatory molecules as compared to other cocktails, yet still significantly lower than that induced by conventional PPD. Interestingly, inclusion of the synthetic Mycobacterium tuberculosis 19-kDa lipoprotein (Pam3Cys-SSNKSTTGSGETTTA) as a TLR-stimulant resulted in obvious augmentation of C1-induced pro-inflammatory molecules to levels comparable to that of PPD. In addition, skin testing using sensitized guinea pig model revealed comparable significant reaction to that of conventional PPD. ESAT-6/CPF-10/MPB-83 cocktail is suggested as a potential alternative skin-testing reagent when used in combination with the M. tuberculosis 19-kDa lipoprotein as a TLR-stimulant. [ABSTRACT FROM AUTHOR]

Paratuberculosis, also known as Johne's disease (JD), is a chronic contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is incurable, fatal and causes economic losses estimated to exceed 200 million dollars to the U.S. dairy industry annually. Several preventive and control measures have been recommended; however, only a few of these measures have been validated empirically. Using a nested compartmental (NC) modeling approach, the main objective of this research was to identify the best combination of control and preventive measures that minimizes the prevalence and incidence of JD and the risk of MAP occurrence in a dairy herd. The NC model employs both MAP transmission estimates and data on pen movement of cattle on a dairy to quantify the effectiveness of control and preventive measures. To obtain reasonable ranges of parameter values for between-pen movements, the NC model was fitted to the movement data of four typical California dairy farms. Using the estimated ranges of the movement parameters and those of JD from previous research, the basic reproduction number was calculated to measure the risk of MAP occurrence in each pen environment as well as the entire dairy. Although the interventions evaluated by the NC model were shown to reduce the infection, no single measure alone was capable of eradicating the infection. The numerical simulations suggest that a combination of test and cull with more frequent manure removal is the most effective method in reducing incidence, prevalence and the risk of MAP occurrence. Other control measures such as limiting calf-adult cow contacts, raising calves in a disease-free herd or colostrum management were less effective. [ABSTRACT FROM AUTHOR]

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world’s population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori. [ABSTRACT FROM AUTHOR]

The global spread of bacterial resistance to drugs used in therapy requires new potent and safe antimicrobial agents. DNA gyrases represent important targets in drug discovery. Schiff bases, thiazole, and triazole derivatives are considered key scaffolds in medicinal chemistry. Fifteen thiazolyl-triazole Schiff bases were evaluated for their antibacterial activity, measuring the growth inhibition zone diameter, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC), against Gram-positive (Staphylococcus aureus, Listeria monocytogenes) and Gram-negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa) bacteria. The inhibition of S. aureus and S. typhimurium was modest. Compounds B1, B2, and B9 showed a similar effect as ciprofloxacin, the antimicrobial reference, against L. monocytogenes. B10 displayed a better effect. Derivatives B1, B5-7, B9, and B11-15 expressed MIC values lower than the reference, against L. monocytogenes. B5, B6, and B11-15 strongly inhibited the growth of P. aeruginosa. All compounds were subjected to an in silico screening of the ADMET (absorption, distribution, metabolism, elimination, toxicity) properties. Molecular docking was performed on the gyrA and gyrB from L. monocytogenes. The virtual screening concluded that thiazolyl-triazole Schiff base B8 is the best drug-like candidate, satisfying requirements for both safety and efficacy, being more potent against the bacterial gyrA than ciprofloxacin. [ABSTRACT FROM AUTHOR]

Culture based phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis (TB) is time consuming therefore rapid genotypic methods are increasingly being utilized. We previously developed and evaluated on TB isolates a rapid genotypic TaqMan array card (TAC) that detects mutations in several resistance-associated genes using dozens of primer pairs, probes, and high resolution melt analysis, with >96% accuracy versus Sanger sequencing. In this study we examined the performance of TAC on sputum, comparing results between 71 paired sputum and TB isolates of which 62 were MDR-TB. We also adapted the TAC to include wild-type probes and broadened coverage for rpoB and gyrA mutations. TAC was 89% successful at detecting wild-type or mutations within inhA, katG, rpoB, eis, gyrA, rplC, and pncA on smear positive sputa and 33% successful on smear negative sputa. The overall accuracy of these detections as compared to the TAC results of the paired isolate was 95% ± 7 (average sensitivity 98% ± 3; specificity 92% ± 14). Accuracy of sputum TAC results versus phenotypic DST for isoniazid, rifampin, ofloxacin/moxifloxacin, and pyrazinamide was 85% ± 12. This was similar to that of the isolate TAC results (accuracy 88% ± 13), thus inaccuracies primarily reflected intrinsic genotypic-phenotypic discordance. The TAC is a rapid, modular, comprehensive, and accurate TB DST for the major first and second line TB drugs and could be used for supplemental testing of GeneXpert resistant smear positive sputum. [ABSTRACT FROM AUTHOR]

Background: Diagnosing tuberculosis (TB) in farmed red deer (Cervus elaphus) is challenging and might require combining cellular and humoral diagnostic tests. Repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might sensitize or desensitize the subjects to both kinds of diagnostic tools. We evaluated the effect of repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA responsiveness in farmed red deer hinds from a TB-free herd. Eighteen 8-month old hinds were inoculated with bovine and avian PPDs and the mitogen phytohaemagglutinin (PHA), as positive control and concurrently tested by ELISA for antibodies against avian (avian PPD, aPPD and protoplasmatic antigen 3, PPA3) and bovine antigens (bPPD and MPB70). Blood serum was also sampled three weeks after each skin testing round and tested for antibodies against aPPD and bPPD, in order to detect eventual antibody level boosts. Testing took place every six months from winter 2012 until winter 2015. Results: The skin test response to both PPDs peaked during the second and third test round, returning to standard values thereafter. Individual variability was particularly high at the first year and early second year testing rounds (first intradermal test and blood sampling; first winter). The antibody response to avian antigens increased through time, while no such increase was recorded for bovine antigens. The antibody boost three weeks after skin testing was more marked for avian PPD. However, there was no consistent trend in the boosting response through time. Conclusion: Repeated comparative skin testing at six month intervals did not cause progressive increments in skin test responsiveness or antibody production. Specifically, we observed no loss of the skin test response to bPPD and also no progressive loss of the boosting effect in the ELISA responses. However, we recorded increases through time in the antibody levels against avian mycobacterial antigens, possibly due to the progressive exposure to MAP or to other cross-reacting environmental mycobacteria. These findings should be taken into account in designing and interpreting TB testing schemes in farmed deer. [ABSTRACT FROM AUTHOR]

Paratuberculosis (PTB), a chronic granulomatous enteritis produced by Mycobacterium avium subspecies paratuberculosis (MAP), is considered as one of the diseases with the highest economic impact in the ruminant industry. Vaccination against MAP is recommended during the first months after birth on the basis that protection would be conferred before the first contact with mycobacteria. However, little is known about the therapeutic effect of MAP vaccination in controlled experimental conditions. The current study was designed to evaluate the efficacy of vaccination before and after challenge with MAP in a rabbit infection model. The rabbits were divided into four groups: non-infected control (NIC, n = 4), infected control challenged with MAP (IC, n = 5), vaccinated and challenged 1 month after with MAP (VSI, n = 5) and challenged with MAP and vaccinated 2 months later (IVS, n = 5). The results from this study show a quick increase in IFN-γ release upon stimulation with bovine, avian and johnin PPD in animals vaccinated before MAP challenge. All vaccinated animals show an increased humoral response as seen by western blot and ELISA. The final bacteriology index (considering tissue culture and qPCR) shows that the IC group was the most affected. Vaccination after infection (IVS) produced the lowest bacteriology index showing significant differences with the IC group (p = 0.034). In conclusion, vaccination against MAP shows positive effects in a rabbit model. However, vaccination after infection shows a slightly stronger protective effect compared to vaccination before infection, suggesting a therapeutic effect. This feature could be applied to previously infected adult animals under field conditions. [ABSTRACT FROM AUTHOR]

Johne’s disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. [ABSTRACT FROM AUTHOR]

Paratuberculosis is an infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of Crohn's disease patients.Since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. However, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. The prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.In the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 MAP proteins. These proteins were chosen according to a previously published immunoproteomics approach. For each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both B-cells and/or class I MHC antigens. The retrieved peptide sequences were analyzed for their specificity and cross-reactivity. The final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations. [ABSTRACT FROM AUTHOR]

Johne's disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is a chronic infection characterized by the development of granulomatous enteritis in wild and domesticated ruminants. It is one of the most significant livestock diseases not only in the USA but also globally, accounting for USD 200–500 million losses annually for the USA alone with potential link to cases of Crohn's disease in humans. Developing safe and protective vaccines is of a paramount importance for JD control in dairy cows. The current study evaluated the safety, immunity and protective efficacy of a novel live attenuated vaccine (LAV) candidate with and without an adjuvant in comparison to an inactivated vaccine. Results indicated that the LAV, irrespective of the adjuvant presence, induced robust T cell immune responses indicated by proinflammatory cytokine production such as IFN-γ, IFN-α, TNF-α and IL-17 as well as strong response to intradermal skin test against M. paratuberculosis antigens. Furthermore, the LAV was safe with minimal tissue pathology. Finally, calves vaccinated with adjuvanted LAV did not shed M. paratuberculosis post-challenge, a much-desired characteristic of an effective vaccine against JD. Together, this data suggests a strong potential of testing LAV in field trials to curb JD in dairy herds. [ABSTRACT FROM AUTHOR]